Chan Woo Song

Bio-based production of C2-C6 platform chemicals.

Platform chemicals composed of 2–6 carbons derived from fossil resources are used as important precursors for making a variety of chemicals and materials, including solvents, fuels, polymers, pharmaceuticals, perfumes, and foods. Due to concerns regarding our environment and the limited nature of fossil resources, however, increasing interest has focused on the development of sustainable technologies for producing these platform chemicals from renewable resources. The techniques and strategies for developing microbial strains for chemicals production have advanced rapidly, and it is becoming feasible to develop microbes for producing additional types of chemicals, including non‐natural molecules. In this study, we review the current status of the bio‐based production of major C2–C6 platform chemicals, focusing on the microbial production of platform chemicals that have been used for the production of chemical intermediates, building block compounds, and polymers. Biotechnol. Bioeng. 2012; 109: 2437–2459.

Biorefineries for the production of top building block chemicals and their derivatives

Due to the growing concerns on the climate change and sustainability on petrochemical resources, DOE selected and announced the bio-based top 12 building blocks and discussed the needs for developing biorefinery technologies to replace the current petroleum based industry in 2004. Over the last 10 years after its announcement, many studies have been performed for the development of efficient technologies for the bio-based production of these chemicals and derivatives. Now, ten chemicals among these top 12 chemicals, excluding the l-aspartic acid and 3-hydroxybutyrolactone, have already been commercialized or are close to commercialization. In this paper, we review the current status of biorefinery development for the production of these platform chemicals and their derivatives. In addition, current technological advances on industrial strain development for the production of platform chemicals using micro-organisms will be covered in detail with case studies on succinic acid and 3-hydroxypropionic acid as examples.

Metabolic engineering of Escherichia coli for the production of fumaric acid

Fumaric acid is a naturally occurring organic acid that is an intermediate of the tricarboxylic acid cycle. Fungal species belonging to Rhizopus have traditionally been employed for the production of fumaric acid. In this study, Escherichia coli was metabolically engineered for the production of fumaric acid under aerobic condition. For the aerobic production of fumaric acid, the iclR gene was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the fumA, fumB, and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. Based on in silico flux response analysis, this base strain was further engineered by plasmid‐based overexpression of the native ppc gene, encoding phosphoenolpyruvate carboxylase (PPC), from the strong tac promoter, which resulted in the production of 4.09 g/L of fumaric acid. Additionally, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to block the conversion of fumaric acid into L‐aspartic acid. Since it is desirable to avoid the use of inducer, the lacI gene was also deleted. To increase glucose uptake rate and fumaric acid productivity, the native promoter of the galP gene was replaced with the strong trc promoter. Fed‐batch culture of the final strain CWF812 allowed production of 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of 0.389 g fumaric acid/g glucose and 0.448 g/L/h, respectively. This study demonstrates the possibility for the efficient production of fumaric acid by metabolically engineered E. coli. Biotechnol. Bioeng. 2013; 110: 2025–2034.

Genome engineering and gene expression control for bacterial strain development

In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron‐mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high‐throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen.

Rapid one‐step inactivation of single or multiple genes in Escherichia coli

Gene knockout experiments are frequently performed for both fundamental and applied biological research. We developed an integration helper plasmid‐based knockout system for more efficient and rapid engineering of Escherichia coli. The integration helper plasmid, pCW611, contains two recombinases that are expressed in the reverse direction by two independent inducible systems. One is Red recombinase under the control of the arabinose‐inducible system to induce a recombination event by using the linear gene knockout DNA fragment, while the other is Cre recombinase, which is controlled by the isopropyl β‐D‐1‐thiogalactopyranoside‐inducible system to obtain markerless mutant strains. The time and effort required can be reduced with this system because iterative transformation and curing steps are not required. We could delete one target gene in three days by using pCW611. To verify the usefulness of this system, deletion experiments were performed to knock out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE–aspA and sfcA–aspA). Also, sequential deletion of four target genes (fumB, iclR, fumA, and fumC) was successfully performed to make a fumaric acid producing strain. This successfully developed and validated rapid and efficient gene manipulation system should be useful for the metabolic engineering of E. coli.

Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid.

A novel metabolic pathway was designed for the production of 3-aminopropionic acid (3-AP), an important platform chemical for manufacturing acrylamide and acrylonitrile. Using a fumaric acid producing Escherichia coli strain as a host, the Corynebacterium glutamicum panD gene (encoding L-aspartate-α-decarboxylase) was overexpressed and the native promoter of the aspA gene was replaced with the strong trc promoter, which allowed aspartic acid production through the aspartase-catalyzed reaction. Additional overexpression of aspA and ppc genes, and supplementation of ammonium sulfate in the medium allowed production of 3.49 g/L 3-AP. The 3-AP titer was further increased to 3.94 g/L by optimizing the expression level of PPC using synthetic promoters and RBS sequences. Finally, native promoter of the acs gene was replaced with strong trc promoter to reduce acetic acid accumulation. Fed-batch culture of the final strain allowed production of 32.3 g/L 3-AP in 39 h.